Muscular atrophy-inducing agent using hypometabolism-inducing substance t1am, and use thereof in treating muscular hypertrophy

ABSTRACT

A screening method for a drug for treating muscular atrophy is proposed. The method may include the steps of: (a) inducing muscular atrophy by administering to a normal cell or a normal animal a hypometabolism-inducing substance selected from the group consisting of 3-iodothyronamine (T1AM), [D-Ala2, D-Leu5] enkephalin (DADLE), 5′-adenosine monophosphate (5′-AMP), and hydrogen sulfide (H2S); (b) treating a candidate substance in the cell or animal treated with the hypometabolism-inducing substance; (c) evaluating the degree of improvement or treatment of muscular atrophy in the cell or animal treated with the candidate substance; and (d) determining the candidate substance as a drug for treating muscular atrophy.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.15/576,231, filed Nov. 21, 2017, which is a national stage applicationof PCT/KR2016/006955, filed Jun. 29, 2016, which claims priority to KR10-2015-0091807, filed Jun. 29, 2015 and KR 10-2016-0068882, filed Jun.2, 2016, the entire disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION Technical Field

The present invention relates to a muscular atrophy-inducing agent basedon hypometabolism efficacy of 3-iodothyronamine (T1AM) and a compositionfor preventing or treating muscular hypertrophy including myotoniacongenital, calf hypertrophy, myhre syndrome, and myostatin-relatedmuscular hypertrophy, or for facial muscle shrinkage using thehypometabolism-inducing substance.

Description of Related Art

The muscular atrophy occurs in various pathological and physiologicalconditions such as bodily injury, cancer cachexia in cancer patients,muscle aging, long-term bed life, or space flight as well as geneticdisorders (e.g., Duchenne muscle dystrophy). Amounts of muscle proteinssuch as actin and myosin are decreased, and muscle mass and musclestrength are significantly decreased. Accordingly, since the muscularatrophy has an effect on most of activities from simple behaviors toroutine tasks, exercises, and even astronaut missions, a pharmacologicalrehabilitative medical research to treat the muscular atrophy isimportant.

A first step for treating of the muscular atrophy is to develop anappropriate model to induce the muscular atrophy. As an animal model (invivo), denervation and hindlimb suspension methods have been mainlyused. Treatment methods of dexamethasone which is syntheticglucocorticoid, oxidizing substances (for example, active oxygen such asH₂O₂), or the like have been used as drugs. It has been known that theanimal model and the drugs activate signaling pathways which areassociated with muscle protein catabolism such as activation of forkheadbox O (FoxO), increased expression of ubiquitin E3 ligase and proteasomewithout exception and simultaneously, inhibit signaling pathways(Akt1-S6K) which are associated with muscle protein anabolism (Shimizuet al., 2011). Further, it has been known that the expression ofchaperone proteins (e.g., heat shock proteins) that help proteinbiogenesis, repair of damage, and the like during the muscular atrophyis also decreased (Gwag et al. 2009).

When describing the mechanism of the muscular atrophy-inducing agents,according to recent reports, it is known that the dexamethasone as asteroid hormone-based substance having an anti-inflammatory effect bindsto a glucocorticoid receptor (GR) and activates a proteolytic signalingpathway of FoxO-proteasome to induce the muscular atrophy. It isreported that oxidative substances such as hydrogen peroxide damage thesarcoplasmic reticulum membrane and the mitochondrial membrane, and thereleased Ca²⁺ and cytochrome C accelerate the activation of calpainproteases to induce the muscular atrophy (McClung et al. 2009).

In addition, the muscular hypertrophy is a disease caused when thebalance of muscle protein synthesis and degradation breaks down, and astypical examples, there are myotonia congenita, calf hypertrophy, myhresyndrome, myostatin-related muscular hypertrophy, and the like. Amongthese diseases, particularly, the myostatin-related muscular hypertrophyis a symptom caused by breakdown of a myostatin gene associated with themuscle protein degradation. Myostatin serves to inhibit a muscle proteinsynthesis pathway (e.g., Akt1-mTOR) and increases the activity of amuscle protein degradation pathway (e.g., SMAD-proteasome), but whenthis gene is broken, the balance of muscle mass retention is broken andthus the muscular hypertrophy occurs.

Meanwhile, 3-iodothyronamine (T1AM) is a derivative of thyroid hormonesT3 and T4 and a hypometabolism-inducing substance that may be generatedin the body. It has been found that a pico mole of 3-iodothyronamine ispresent in most of the rodent tissue samples (brain, liver, heart,kidney, muscle, etc.) and the human blood (Zucchi R et al., 2006). Inaddition, 3-iodotronamine is a synthesizable substance and a preparingmethod thereof is disclosed in U.S. Pat. Nos. 6,979,750 and 7,321,065and Korean Patent Registration No. 1,112,731, which is a prior patent ofthe inventor of the present application, and the 3-iodotronamine can bemass-produced to be easily used for industrial use.

The present inventors found that muscular atrophy may be induced bytreating a hypometabolism-inducing substance according to proteinexpression levels associated with generation and inhibition of muscleproteins and a change in size of myotube cells and intend to provide anew concept of muscular atrophy study model which is different fromexisting methods using the hypometabolism-inducing substance, and amuscle hyperthrophy treating agent through a muscular atrophy inhibitioneffect or a composition for facial muscle shrinkage usable for Botox.

BRIEF SUMMARY OF THE INVENTION

An object of the present invention is to provide a muscular atrophystudy model as a novel muscular atrophy inducing method which isdifferent from a denervation method, a hindlimb suspension method, and amethod for treating dexamethasone as synthetic glucorticoid, anoxidizing agent (e.g., active oxygen such as H₂O₂), or the like in therelated art. Herein, the study model may include cell, tissue, andanimal models.

Another object of the present invention is to provide a pharmaceuticalcomposition or a health food for preventing or treating muscularhypertrophy using a hypometabolism-inducing substance of the presentinvention.

Yet another object of the present invention is to provide a compositionfor facial muscle shrinkage usable for Botox using ahypometabolism-inducing substance of the present invention.

In order to achieve the objects, an exemplary embodiment of the presentinvention provides a muscular atrophy-inducing agent containing as anactive ingredient a hypometabolism-inducing substance selected from thegroup consisting of 3-iodothyronamine (T1AM), [D-Ala2,D-Leu5] enkephalin(DADLE), 5′-adenosine monophosphate (5′-AMP), and hydrogen sulfide(H₂S), by targeting an animal model, a muscular atrophy study modelincluding inducing muscular atrophy by administering the muscularatrophy-inducing agent to cells and animals, and a drug screening methodfor preventing or treating muscular atrophy based on the muscularatrophy study model.

Another exemplary embodiment of the present invention provides apharmaceutical composition for preventing or treating muscularhypertrophy using the hypometabolism-inducing substance.

Yet another exemplary embodiment of the present invention provides ahealth food for preventing or treating muscular hypertrophy using thehypometabolism-inducing substance.

Still another exemplary embodiment of the present invention provides acomposition for facial muscle shrinkage using thehypometabolism-inducing substance.

According to the present invention, the muscular atrophy inducing modelmay provide an economic muscular atrophy study model by using ahypometabolic compound enabling mass production and may be usefully usedby verification for screening a drug for preventing or treating muscularatrophy. Further, since the hypometabolic compound significantlyactivates muscle protein degradation, the hypometabolic compound may beusefully used as a drug for treating muscular hypertrophy or acomposition for facial muscle shrinkage.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1B illustrate comparison of diameters of C2C12 myotubes betweena T1AM treated group and a control: FIG. 1A illustrates a representativephotograph of myotubes taken with an Axiovert 200 optical microscope(magnification: 200×0, in which a pair of arrows indicates locationswhere diameters of the myotubes are measured (black bar=25 μm). FIG. 1Bis a graph illustrated by measuring the diameters of myotubes betweentwo groups. Data: Mean±SEM (n=3; 96 cells/group), *: It means that thereis a significant difference between the two groups [independent samplest-test, P<0.05)].

FIGS. 2A-2C illustrate comparison of AMPK activities of C2C12 myotubesbetween a T1AM treated group and a control: FIG. 2A illustrates a resultof Immunoblotting analysis for expression of p-AMPK and AMPK. FIG. 2B isa graph illustrating expression levels of p-AMPK and AMPK asdensitometric quantitation, and FIG. 2C is a graph illustrating anexpression ratio of p-AMPK/AMPK as densitometric quantitation [mean±SEM(n=6), *, P<0.05].

FIGS. 3A-3E illustrates comparison of expression of Akt1 and S6K ofC2C12 myotubes between a T1AM treated group and a control: FIG. 3Aillustrates a result of Immunoblotting analysis for expression of Akt1and S6K. FIGS. 3B and 3C are graphs illustrating expression levels ofp-Akt1 and Akt1 and an expression ratio of p-Akt1/Akt1, respectively.FIGS. 3D and 3E are graphs illustrating expression levels of p-S6K andS6K and an expression ratio of p-S6K/S6K, respectively [mean±SEM (n =6),*, P<0.05].

FIGS. 4A-4F illustrate comparison of expression of FoxO1 and FoxO3 ofC2C12 myotubes between a T1AM treated group and a control: FIG. 4Aillustrates a result of immunoblotting analysis for expression of FoxO1and FoxO3. FIG. 4B is a photograph taken as an analysis result ofimmunofluorescence staining for FoxO1 and FoxO3 with a confocalmicroscope. FIGS. 4C and FIG. 4D are graphs illustrating expressionlevels of p-FoxO1 and FoxO1 and an expression ratio of p-FoxO1/FoxO1,respectively. FIG. 4E and FIG. 4F are graphs illustrating expressionlevels of p-FoxO1 and FoxO1 and an expression ratio of p-FoxO3/FoxO3,respectively [mean±SEM (n=6), *, P<0.05].

FIGS. 5A-5D illustrates comparison of expression of MuRF1 and MAFbx ofC2C12 myotubes between a T1AM treated group and a control: A illustratesa result of immunoblotting analysis for expression of FoxO1 and FoxO3.FIG. 5B and FIG. 5C are graphs illustrating densitometric quantitationfor expression levels of MuRF1 and MAFbx. FIG. 5D illustrateschymotrypsin-like activity of 26S, which is determined throughcell-based luminescence analysis and expressed as a relative light unit(RLU). Actual chymotrypsin-like activity was determined from <totalRLUs—background RLUs>in each analysis [mean±SEM (n=6), *, P<0.05].

FIGS. 6A-6D illustrates comparison of expression of chaperone of C2C12myotubes between a T1AM treated group and a control: A illustrates aresult of immunoblotting analysis of expression of heat shock protein 72(HSP72), HSP60 and αB-crystallin. FIG. 6B and FIG. 6D are graphsillustrating densitometric quantitation for an expression level of eachchaperone protein [mean±SEM (n=6), *, P<0.05].

FIG. 7 is a schematic diagram of signaling pathways associated withsynthesis and degradation of muscle proteins according to T lAMtreatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a muscular atrophy-inducing agentcontaining as an active ingredient a hypometabolism-inducing substanceselected from the group consisting of 3-iodothyronamine (T1AM),[D-Ala2,D-Leu5] enkephalin (DADLE), 5′-adenosine monophosphate (5′-AMP),and hydrogen sulfide (H₂S), by targeting an animal model, a method forpreparing a muscular atrophy study model including inducing muscularatrophy by treating or administering the muscular atrophy-inducingagent, a sturdy model prepared according to the method, a method forusing drug screening for preventing or treating muscular atrophy usingthe muscular atrophy study model, and a for preventing or treatingmuscular hypertrophy using the hypometabolic substance as an activeingredient or a composition for facial muscle shrinkage using thehypometabolic substance as an active ingredient. The present inventorsfound the fact that the hypometabolism-inducing substance inhibited amuscle protein synthesis mechanism and activated a degradation mechanismthrough related protein expression and a change in size of myotubes andcompleted the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides a muscular atrophy-inducing agentcontaining as an active ingredient a hypometabolism-inducing substanceselected from the group consisting of 3-iodothyronamine (T1AM),[D-Ala2,D-Leu5] enkephalin (DADLE), 5′-adenosine monophosphate (5′-AMP),and hydrogen sulfide (H₂S).

In one embodiment of the present invention, the hypometabolism-inducingsubstance may be more particularly 3-iodothyronamine (T1AM).

In one embodiment of the present invention, in the study model, as acell line, a muscle cell line may be used, general muscle cell lines ormuscle fibers used in the art may be used, and for example, C2C12 musclecells and the like may be used.

In one embodiment of the present invention, in the study model, theanimal may be vertebrate animals, more specifically vertebrate animalsexcept for humans, and for example, may include rodents including mice,rats, and hamsters, rabbits, horses, cows, dogs, cats, monkeys, guineapigs, and the like.

Further, the present invention provides a method for preparing amuscular atrophy study model including inducing muscular atrophy byadministering to a normal animal a hypometabolism-inducing substanceselected from the group consisting of 3-iodothyronamine (T1AM),[D-Ala2,D-Leu5] enkephalin (DADLE), 5′-adenosine monophosphate (5′-AMP),and hydrogen sulfide (H₂S).

In one embodiment of the present invention, the hypometabolism-inducingsubstance may be more particularly 3-iodothyronamine (T1AM), and a doseof the T1AM may be appropriately adjusted. For example, when T1AM isadministered intraperitoneally, if the dose of T1AM is less than 10mg/kg per unit weight (kg) of an administered animal, it is difficult tocause muscular atrophy, and if the dose exceeds 500 mg/kg, the animaldies. As a result, the close of T1AM may be 10 to 500 mg/kg, morespecifically 20 to 250 mg/kg, and more specifically 25 to 100 mg/kg, perunit body weight (kg) of animal, but may be appropriately adjustedaccording to the condition of the animal and experimental conditions.

In one embodiment of the present invention, the treatment concentrationin the cells may be 0.1 μM to 1000 μM, but may be appropriately adjustedaccording to the amount of cells, conditions of the cells, andexperimental conditions.

In one embodiment of the present invention, the administration of thehypometabolism-inducing substance may be performed by a generaladministration method, such as oral administration, intraperitonealadministration, intravenous administration, intramuscularadministration, subcutaneous administration, or intradermaladministration, but the administration method is not limited thereto.Further, the hypometabolism-inducing substance may be administered byany device which is movable to a target cell as an active substance.

In one embodiment of the present invention, the number of dose times ofthe hypometabolism-inducing substance may be one to two or more timesper day, but the number of dose times may be controlled according to thedose of the hypometabolism-inducing substance.

In one embodiment of the present invention, the degree of muscularatrophy in the muscular atrophy animal model may be adjusted bycontrolling a dose of the hypometabolism-inducing substance or anexposure time in vivo of the hypometabolism-inducing substance, and thismay be performed based on the degree of muscular atrophy increasing inproportion to the dose or the exposure time in vivo.

Further, the present invention provides a muscular atrophy cell model oranimal model prepared by the method.

In one embodiment of the present invention, the muscular atrophy studymodel may show the following results from (a) to (j) as compared to anormal by the administration of hypometabolism-inducing substance, whichwas confirmed by a muscle cell experiment:

(a) Decrease in size of myotubes or size of muscle;

(b) Increase in expression ratio of p-AMPK/AMPK;

(c) Decrease in expression ratio of p-Akt1/Akt1;

(d) Decrease in expression ratio of p-S6K/S6K;

(e) Decrease in expression ratio of p-FoxO1/FoxO1;

(f) Decrease in expression ratio of p-FoxO3/FoxO3;

(g) Increase in expression level of MuRF1;

(h) Increase in activity of proteasome;

(i) Decrease in expression level of heat shock protein 72 (HSP72); and

(j) Decrease in expression level of αB-crystallin

Accordingly, the muscular atrophy study model may have at least onecharacteristic selected from the group consisting of (a) a decrease insize of myotubes or size of muscle; (b) an increase in expression ratioof p-AMPK/AMPK; (c) a decrease in expression ratio of p-Akt1/Akt1; (d) adecrease in expression ratio of p-S6K/S6K; (e) a decrease in expressionratio of p-FoxO1/FoxO1; (f) a decrease in expression ratio ofp-FoxO3/FoxO3; (g) an increase in expression level of MuRF1; (h) anincrease in activity of proteasome; (i) a decrease in expression levelof heat shock protein 72 (HSP72); and (j) a decrease in expression levelof αB-crystallin, as compared to a normal.

In one embodiment of the present invention, (a) to (j) may becharacteristics compared to the normal after 5 to 10 days ofadministration of the hypometabolism-inducing substance.

In one embodiment of the present invention, when 0.1 μM to 1000 μM ofT1MA as the hypometabolism-inducing substance is treated to themyotubes, with respect to (a) above, the size of the myotubes may bedecreased by 0.01 to 0.20 times as compared to the size of the normalmyotubes. With respect to (b) above, the expression ratio of p-AMPK/AMPKin the muscular atrophy study model may be increased by 0.01 to 2.5times as compared to the expression ratio of p-AMPK/AMPK in the normalmodel. With respect to (c) above, the expression ratio of p-Akt1/Akt1 inthe muscular atrophy study model may be decreased by 0.01 to 1.0 timesas compared to the expression ratio of p-Akt1/Akt1 in the normal model.With respect to (d) above, the expression ratio of p-S6K/S6K in themuscular atrophy study model may be decreased by 0.01 to 1.0 times ascompared to the expression ratio of p-S6K/S6K in the normal model. Withrespect to (e) above, the expression ratio of p-FoxO1/FoxO1 in themuscular atrophy study model may be decreased by 0.01 to 1.0 times ascompared to the expression ratio of p-FoxO1/FoxO1 in the normal model.With respect to (f) above, the expression ratio of p-FoxO3/FoxO3 in themuscular atrophy study model may be decreased by 0.01 to 0.8 times ascompared to the expression ratio of p-FoxO3/FoxO3 in the normal model.With respect to (g) above, the expression level of MuRF1 in the muscularatrophy study model may be increased by 0.01 to 2.5 times as compared tothe expression level of MuRF1 in the normal model. With respect to (h)above, the activity of proteasome in the muscular atrophy study modelmay be increased by 0.01 to 2.0 times as compared to the activity ofproteasome in the normal model. The proteasome may be 26S proteasome,and the activity thereof can be measured using a method known as amethod for measuring the activity of proteasome. For example, theactivity of proteasome may be analyzed through chemotrypsin-likeactivity. With respect to (i) above, the expression level of heat shockprotein 72 (HSP72) in the muscular atrophy study model may be increasedby 0.01 to 0.15 times as compared to the expression level of heat shockprotein 72 (HSP72) in the normal model. With respect to (j) above, theexpression level of αB-crystallin in the muscular atrophy study modelmay be decreased by 0.01 to 1.0 times as compared to the expressionlevel of αB-crystallin in the normal model. The expression levels ofAMPK, phospho-AMPK (p-AMPK), FoxO1, p-FoxO1, FoxO3, p-FoxO3, Akt1,p-Akt1, S6K, p-S6K, MuRF1, HSP72, and αB-crystallin in (b) to (g), (i),and (j) may be measured by using methods known as a protein analysismethod. For example, the expression levels may be measured by animmunoblotting method.

The muscular atrophy study model may be used as a study model foraccurate muscular atrophy studies, but may also be useful asverification for screening of a drug of preventing or treating muscularatrophy.

Accordingly, the present invention provides a method for screening adrug for treating muscular atrophy including: treating a candidatesubstance in the muscular atrophy study model;

and determining the candidate substance as the drug for treatingmuscular atrophy by evaluating the improved or treated degree of themuscular atrophy in the sturdy model treated with the candidatesubstance.

In one embodiment of the present invention, the candidate substance is asubstance capable of treating muscular atrophy and includes chemicals,oligonucleotides, peptides, genes, proteins, and the like, withoutlimitation.

In one embodiment of the present invention, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing at least oneof the following indicators (1) to (10) with that of the control:

(1) Size of myotubes or size of muscle;

(2) Expression ratio of p-AMPK/AMPK;

(3) Expression ratio of p-Akt1/Akt1;

(4) Expression ratio of p-S6K/S6K;

(5) Expression ratio of p-FoxO1/FoxO1;

(6) Expression ratio of p-FoxO3/FoxO3;

(7) Expression level of MuRF1;

(8) Activity of proteasome;

(9) Expression level of heat shock protein 72 (HSP72); and

(10) Expression level of αB-crystallin

With respect to indicator (1) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing changes inthe size of the myotubes or the size of the muscle in the group treatedwith the candidate substance and the control. When the size of themyotubes or the muscle in the group treated with the candidate substanceis increased compared to that of the control, the candidate substancemay be determined as a drug for treating muscular atrophy.

With respect to indicator (2) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing expressionratios of p-AMPK/AMPK in the group treated with the candidate substanceand the control. When the expression ratio of p-AMPK/AMPK in the grouptreated with the candidate substance is decreased compared to that ofthe control, the candidate substance may be determined as a drug fortreating muscular atrophy.

With respect to indicator (3) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing expressionratios of p-Akt1/Akt1 in the group treated with the candidate substanceand the control. When the expression ratio of p-Akt1/Akt1 in the grouptreated with the candidate substance is increased compared to that ofthe control, the candidate substance may be determined as a drug fortreating muscular atrophy.

With respect to indicator (4) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing expressionratios of p-S6K/S6K in the group treated with the candidate substanceand the control. When the expression ratio of p-S6K/S6K in the grouptreated with the candidate substance is increased compared to that ofthe control, the candidate substance may be determined as a drug fortreating muscular atrophy.

With respect to indicator (5) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing expressionratios of p-FoxO1/FoxO1 in the group treated with the candidatesubstance and the control. When the expression ratio of p-FoxO1/FoxO1 inthe group treated with the candidate substance is increased compared tothat of the control, the candidate substance may be determined as a drugfor treating muscular atrophy.

With respect to indicator (6) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing expressionratios of p-FoxO3/FoxO3 in the group treated with the candidatesubstance and the control. When the expression ratio of p-FoxO3/FoxO3 inthe group treated with the candidate substance is increased compared tothat of the control, the candidate substance may be determined as a drugfor treating muscular atrophy.

With respect to indicator (7) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing expressionlevels of MuRF1 in the group treated with the candidate substance andthe control. When the expression level of MuRF1 in the group treatedwith the candidate substance is decreased compared to that of thecontrol, the candidate substance may be determined as a drug fortreating muscular atrophy.

With respect to indicator (8) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing proteasomeactivities in the group treated with the candidate substance and thecontrol. When the activity of proteasome in the group treated with thecandidate substance is decreased compared to that of the control, thecandidate substance may be determined as a drug for treating muscularatrophy.

With respect to indicator (9) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing expressionlevels of hot shock protein 72 (HSP72) in the group treated with thecandidate substance and the control. When the expression level of hotshock protein 72 (HSP72) in the group treated with the candidatesubstance is increased compared to that of the control, the candidatesubstance may be determined as a drug for treating muscular atrophy.

With respect to indicator (10) above, the degree of improvement ortreatment of muscular atrophy may be evaluated by comparing expressionlevels of αB-crystallin in the group treated with the candidatesubstance and the control. When the expression level of αB-crystallin inthe group treated with the candidate substance is increased compared tothat of the control, the candidate substance may be determined as a drugfor treating muscular atrophy.

Accordingly, in the determining, when the group treated with thecandidate substance is compared to the control, in the case of at leastone result selected from the group consisting of an increase in size ofmyotubes or size of muscle; a decrease in expression ratio ofp-AMPK/AMPK; an increase in expression ratio of p-Akt1/Akt1; an increasein expression ratio of p-S6K/S6K; an increase in expression ratio ofp-FoxO1/FoxO1; an increase in expression ratio of p-FoxO3/FoxO3; adecrease in expression level of MuRF1; a decrease in activity ofproteasome; an increase in expression level of heat shock protein 72(HSP72); and an increase in expression level of αB-crystallin, thecandidate substance may be determined as a drug for treating muscularatrophy.

The control refers to a group treated with an excipient of the drug fortreating muscular atrophy instead of the candidate substance, and forexample, the control may be a group consisting of dimethyl sulfoxide(DMSO), physiological saline, sterilized distilled water, carboxymethylcellulose or phosphate buffered saline (PBS).

The present invention provides a screening method for a drug forpreventing muscular atrophy including: treating or administering acandidate substance to normal cells or a normal animal; treating oradministering a hypometabolism-inducing substance to the cells or theanimal;

and determining the candidate substance as the drug for preventingmuscular atrophy by evaluating the degree of muscular atrophy in thecells or the animal treated with the hypometabolism-inducing substance.

In one embodiment of the present invention, the candidate substance isthe same as described above.

In one embodiment of the present invention, the degree of muscularatrophy may be evaluated by comparing at least one of the followingindicators (1) to (10) with that of the control:

(1) Size of myotubes or size of muscle;

(2) Expression ratio of p-AMPK/AMPK;

(3) Expression ratio of p-Akt1/Akt1;

(4) Expression ratio of p-S6K/S6K;

(5) Expression ratio of p-FoxO1/FoxO1;

(6) Expression ratio of p-FoxO3/FoxO3;

(7) Expression level of MuRF1;

(8) Activity of proteasome;

(9) Expression level of heat shock protein 72 (HSP72); and

(10) Expression level of αB-crystallin

With respect to indicator (1) above, the degree of muscular atrophy maybe evaluated by comparing changes in the size of the myotubes or thesize of the muscle in the group treated with the candidate substance andthe control. When the size of the myotube or the muscle in the grouptreated with the candidate substance is increased compared to that ofthe control, the candidate substance may be determined as a drug forpreventing muscular atrophy.

With respect to indicator (2) above, the degree of muscular atrophy maybe evaluated by comparing expression ratios of p-AMPK/AMPK in the grouptreated with the candidate substance and the control. When theexpression ratio of p-AMPK/AMPK in the group treated with the candidatesubstance is decreased compared to that of the control, the candidatesubstance may be determined as a drug for preventing muscular atrophy.

With respect to indicator (3) above, the degree of muscular atrophy maybe evaluated by comparing expression ratios of p-Akt1/Akt1 in the grouptreated with the candidate substance and the control. When theexpression ratio of p-Akt1/Akt1 in the group treated with the candidatesubstance is increased compared to that of the control, the candidatesubstance may be determined as a drug for preventing muscular atrophy.

With respect to indicator (4) above, the degree of muscular atrophy maybe evaluated by comparing expression ratios of p-S6K/S6K in the grouptreated with the candidate substance and the control. When theexpression ratio of p-S6K/S6K in the group treated with the candidatesubstance is increased compared to that of the control, the candidatesubstance may be determined as a drug for preventing muscular atrophy.

With respect to indicator (5) above, the degree of muscular atrophy maybe evaluated by comparing expression ratios of p-FoxO1/FoxO1 in thegroup treated with the candidate substance and the control. When theexpression ratio of p-FoxO1/FoxO1 in the group treated with thecandidate substance is increased compared to that of the control, thecandidate substance may be determined as a drug for preventing muscularatrophy.

With respect to indicator (6) above, the degree of muscular atrophy maybe evaluated by comparing expression ratios of p-FoxO3/FoxO3 in thegroup treated with the candidate substance and the control. When theexpression ratio of p-FoxO3/FoxO3 in the group treated with thecandidate substance is increased compared to that of the control, thecandidate substance may be determined as a drug for preventing muscularatrophy.

With respect to indicator (7) above, the degree of muscular atrophy maybe evaluated by comparing expression levels of MuRF1 in the grouptreated with the candidate substance and the control. When theexpression level of MuRF1 in the group treated with the candidatesubstance is decreased compared to that of the control, the candidatesubstance may be determined as a drug for preventing muscular atrophy.

With respect to indicator (8) above, the measurement of the activity ofproteasome is the same as that of (h) described above, and the degree ofmuscular atrophy may be evaluated by comparing activities of proteasomein the group treated with the candidate substance and the control. Whenthe activity of proteasome in the group treated with the candidatesubstance is decreased compared to that of the control, the candidatesubstance may be determined as a drug for preventing muscular atrophy.

With respect to indicator (9) above, the degree muscular atrophy may beevaluated by comparing expression levels of hot shock protein 72 (HSP72)in the group treated with the candidate substance and the control. Whenthe expression level of hot shock protein 72 (HSP72) in the grouptreated with the candidate substance is increased compared to that ofthe control, the candidate substance may be determined as a drug forpreventing muscular atrophy.

With respect to indicator (10) above, the degree of muscular atrophy maybe evaluated by comparing expression levels of αB-crystallin in thegroup treated with the candidate substance and the control. When theexpression level of αB-crystallin in the group treated with thecandidate substance is increased compared to that of the control, thecandidate substance may be determined as a drug for preventing muscularatrophy.

The expression levels of AMPK, phospho-AMPK (p-AMPK), FoxO1, p-FoxO1,FoxO3, p-FoxO3, Akt1, p-Akt1, S6K, p-S6K, MuRF1, HSP72, andαB-crystallin proteins The expression levels in (2) to (7), (9), and(10) may be measured using methods known as a protein analysis method.For example, the expression levels may be measured by immunoblotting.

Therefore, in the determining, when the group treated with the candidatesubstance is compared to the control, in the case of at least one resultselected from the group consisting of an increase in size of myotubes orsize of muscle; a decrease in expression ratio of p-AMPK/AMPK; anincrease in expression ratio of p-Akt1/Akt1; an increase in expressionratio of p-S6K/S6K; an increase in expression ratio of p-FoxO1/FoxO1; anincrease in expression ratio of p-FoxO3/FoxO3; a decrease in expressionlevel of MuRF1; a decrease in activity of proteasome; an increase inexpression level of heat shock protein 72 (HSP72); and an increase inexpression level of αB-crystallin, the candidate substance may bedetermined as a drug for preventing muscular atrophy.

The control refers to a group treated with an excipient of the drug forpreventing muscular atrophy instead of the candidate substance, and forexample, the control may be a group consisting of dimethyl sulfoxide(DMSO), physiological saline, sterilized distilled water, carboxymethylcellulose or phosphate buffered saline (PBS).

In the present specification, the normal cells or the normal animalrefer to cells or animals without muscular atrophy. For example, theanimal may be an animal that is the same species as the muscular atrophymodel and does not have muscular atrophy raised in the same or similarenvironment.

The present invention provides a pharmaceutical composition forpreventing or treating muscular hypertrophy, containing as an activeingredient a hypometabolism-inducing substance selected from the groupconsisting of 3-iodothyronamine (T1AM), [D-Ala2,D-Leu5] enkephalin(DADLE), 5′-adenosine monophosphate (5′-AMP), and hydrogen sulfide(H₂S).

In one embodiment of the present invention, the hypometabolism-inducingsubstance may be more particularly 3-iodothyronamine (T1AM).

In one embodiment of the present invention, the muscular hypertrophyincludes myotonia congenital, calf hypertrophy, myhre syndrome, andmyostatin-related muscular hypertrophy.

In one embodiment of the present invention, since thehypometabolism-inducing substance of the present invention inducesmuscular atrophy by inhibiting activity of Akt1-S6K involved in muscleprotein synthesis and activating FoxO-proteasome involved in muscleprotein degradation, the hypometabolism-inducing substance can be usedas a drug which may replace the role of myostatin and may be used fortreatment of various muscle hypertrophies including myostatin-relatedmuscular hypertrophy caused by binding of myostatin.

The present invention includes all of its pharmaceutically acceptablesalt and solvates, hydrates, racemates, or stereoisomers capable ofbeing prepared therefrom as well as the hypometabolism-inducingsubstance of the present invention.

The hypometabolism-inducing substance of the present invention may beused in a form of its pharmaceutically acceptable salt and as the salt,acid additional salts formed by free pharmaceutically acceptable acidare useful. The acid additional salts are obtained from inorganic acidssuch as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid,hydrobromic acid, hydriodic acid, nitrous acid or phosphorous acid andnon-toxic organic acids such as aliphatic mono and dicarboxylate,phenyl-substituted alkanoate, hydroxy alkanoate and alkandioate,aromatic acids, aliphatic and aromatic sulfonic acids. Thepharmaceutically non-toxic salt includes sulfate, fatigue sulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate, mono-hydrogenphosphate, dihydrogen phosphate, meta-phosphate, pyrophosphate chloride,bromide, iodide, fluoride, acetate, propionate succinate, decanoate,caprylate, acrylate, formate, isobutyrate, caprate, heptanoate,propionic oleate, oxalate, malonate, succinate, suberate, sebacate,fumarate, maleate , butyne-1,4-dioate, hexane-1,6-dioate, benzoate,chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate,methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluenesulfonate, chlorobenzene sulfonate, xylene sulfonate, phenylacetate,phenyl propionate, phenyl butyrate, citrate, lactate, hydroxybutyrate,glycollate, maleate, tartrate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid additional salt according to the present invention may beprepared by a general method, for example, dissolving thehypometabolism-inducing substance of the present invention in a largeamount of acid aqueous solution and precipitating the salt by using awater-miscible organic solvent, for example, methanol, ethanol, acetone,or acetonitrile. Further, the salt which is dried or precipitated byevaporating the solvent or a large amount of acid from the mixture mayalso be prepared through suction-filtering.

Further, a pharmaceutically acceptable metal salt may be prepared byusing base. An alkali metal or alkaline earth metal salt is obtained by,for example, dissolving the compound in a large amount of alkali metalhydroxide or alkaline earth metal hydroxide solution and filtering aninsoluble compound salt and then evaporating and drying a filtrate. Inthis case, the metal salt is pharmaceutically suitable to preparesodium, potassium or calcium salts. Further, the silver saltcorresponding thereto is obtained by reacting alkali metal or alkalineearth metal salts with an appropriate silver salt (for example, silvernitrate).

When the composition is formulated, the formulation is prepared by usingdiluents or excipients, such as a filler, an extender, a binding agent,a wetting agent, a disintegrating agent, and a surfactant, which aregenerally used.

A solid formulation for oral administration includes a tablet, a pill, apowder, a granule, a capsule, a troche agent, or the like, and the solidformulation may be prepared by mixing at least one excipient, forexample, starch, calcium carbonate, sucrose or lactose, gelatin, or thelike with at least one hypometabolism-inducing substance of the presentinvention. Further, lubricants such as magnesium stearate talc may beused in addition to simple excipients. A liquid formulation for oraladministration may use a suspension, a solution, an emulsion, a syrup,and the like, and may include various excipients, for example, a wettingagent, a sweetener, an aromatic agent, a preserving agent, and the likein addition to water and liquid paraffin, as simple diluents which arecommonly used.

A formulation for parenteral administration includes a sterile aqueoussolution, a non-aqueous solution, a suspension, an emulsion, alyophilizing agent, a suppository, and the like.

As the non-aqueous solution and the suspension, propylene glycol,polyethylene glycol, vegetable oils such as olive oil, injectable estersuch as ethyl oleate, and the like may be used. As a matter of thesuppository, witepsol, macrogol, tween 61, cacao butter, laurin,glycerol, gelatin, and the like may be used.

The composition according to the present invention is administered witha pharmaceutically effective dose. In the present invention, the“pharmaceutically effective dose” refers to a amount which is sufficientto treat the diseases at a reasonable benefit/risk ratio applicable tomedical treatment, and an effective dose level may be determinedaccording to elements including a kind of disease of the patient, theseverity, activity of a drug, sensitivity to a drug, a time ofadministration, a route of administration, and an emission rate,duration of treatment, and simultaneously used drugs and other elementswell-known in the medical field. The composition of the presentinvention may be administered as an individual therapeutic agent oradministered in combination with other therapeutic agents, sequentiallyor simultaneously administered with existing therapeutic agents, andadministered singly or multiply. It is important to administer an amountcapable of obtaining a maximum effect with a minimal amount without sideeffects by considering the above elements and the amount may be easilydetermined by those skilled in the art.

Particularly, the effective dose of the composition according to thepresent invention may vary according to age, gender, and weight of thepatient, and generally administered by 0.1 mg to 100 mg per weight 1 kg,preferably administered by 0.5 mg to 10 mg daily or every other day, oradministered one to three times per day. However, since the effectivedose may be decreased or increased depending on the route ofadministration, the severity of obesity, gender, weight, age, and thelike, the dose is not limited to the scope of the present invention inany way.

The present invention provides a health food for preventing or treatingmuscular hypertrophy, containing as an active ingredient ahypometabolism-inducing substance selected from the group consisting of3-iodothyronamine (T1AM), [D-Ala2,D-Leu5] enkephalin (DADLE),5′-adenosine monophosphate (5′-AMP), and hydrogen sulfide (H₂S).

In one embodiment of the present invention, since thehypometabolism-inducing substance of the present invention inducesmuscular atrophy by inhibiting activity of Akt1-S6K involved in muscleprotein synthesis and activating FoxO-proteasome involved in muscleprotein degradation, the hypometabolism-inducing substance can be usedas a drug which may replace the role of myostatin and may be used forhealth foods for preventing or improving various muscle hypertrophiesincluding myostatin-related muscular hypertrophy caused by binding ofmyostatin.

Kinds of foods which are added with the hypometabolism-inducingsubstance of the present invention are not particularly limited.Examples of the foods which may be added with the materials includedrinks, meat, sausages, bread, biscuits, rice cakes, chocolate, candies,snacks, cookies, pizza, ramen noodles, other noodles, gums, dairyproducts including ice cream, various soups, beverages, alcohol drinks,vitamin complex, milk products, milk dairy products, and the like, andinclude all health functional foods in the accepted meaning.

The hypometabolism-inducing substance of the present invention may beadded to the food as it is or may be used together with other food orfood ingredients, and may be appropriately used according to generalmethods. A mixing amount of active ingredients may be appropriatelydetermined according to a purpose of use (for prevention or improvement)thereof. Generally, the amount of compound in the health functional foodmay be added with 0.1 to 90 parts by weight with respect to the entirefood weight. However, in the case of long-term administration for healthand hygiene or health control, the amount may be the range or less.Since there is no problem in terms of safety, the active ingredients maybe used with the amount in the range or more.

In the health food composition according to the present invention, otheringredients are not particularly limited except for containing thecompound as the required ingredient at the indicated ratio, and like ageneral beverage, various flavoring agents, natural starches, or thelike may be contained as an additional ingredient. Examples of theaforementioned natural carbohydrates include general sugars, such asmonosaccharides, for example, glucose, fructose, and the like;disaccharides, for example, maltose, sucrose, and the like; andpolysaccharides, for example, dextrin, cyclodextrin, and the like, andsugar alcohols, such as xylitol, sorbitol, and erythritol. As theflavoring agent other than the above examples, natural flavoring agents(thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)and synthetic flavoring agents (saccharin, aspartame, etc.) may beadvantageously used. A ratio of the natural carbohydrate may begenerally about 1 to 20 g and preferably about 5 to 10 g per 100 g ofthe composition of the present invention.

Further, the health food composition according to the present inventionmay contain various nutrients, vitamins, minerals (electrolytes),flavoring agents such as synthetic flavoring agents and naturalflavoring agents, coloring agents and thickening agents (cheese,chocolate, etc.), pectic acid and salts thereof, alginic acid and saltsthereof, organic acid, a protective colloidal thickener, a pH adjustingagent, a stabilizer, a preservative, glycerin, alcohol, a carbonic acidagent used in a carbonated drink, or the like. Besides, the health foodcomposition of the present invention may include pulps for preparingnatural fruit juice and fruit juice drinks, and vegetable drinks.

These ingredients may be used independently or in combination. The ratioof such additives is not limited, but is generally selected in the rangeof 0.1 to about 20 parts by weight per 100 parts by weight of thehypometabolism-inducing substance of the present invention.

Further, the present invention provides a pharmaceutical composition forfacial muscle shrinkage containing a hypometabolism-inducing substanceof the present invention as an active ingredient.

In one embodiment of the present invention, since thehypometabolism-inducing substance of the present invention inducesmuscular atrophy by inhibiting activity of Akt1-S6K involved in muscleprotein synthesis and activating FoxO-proteasome involved in muscleprotein degradation, the hypometabolism-inducing substance may beusefully used as the composition for facial muscle shrinkage which maybe used for Botox.

Hereinafter, the present invention will be described in more detailthrough Experimental Examples according to the present invention, butthe scope of the present invention is not limited to ExperimentalExamples to be described below and the like.

Experimental Examples 1. Experimental Substances and Method 1) Chemicalsand Storage Solutions

T1AM was chemically synthesized (Korean Patent Registration No.1,112,731) and dissolved in dimethyl sulfoxide (DMSO; SIGMA, Missouri,US) at a storage concentration of 0.75 and 1 M. A DMEM (Welgene,Dalseogu, Daegu, Korea) medium was used, and nonidet P-40, a completemini protease inhibitor, and a phosphatase inhibitor cocktail werepurchased from Roche. A RIPA buffer solution (11% Nonidet P-40, 1%sodium deoxycholate, 150 mM NaCl, 10 mM sodium phosphate [pH 7.4], 2 mMEDTA, 50 mM NaF, 0.2 mM Na₃VO₄, 40 mM HEPES [pH 7.4], 0.7% CHAPS, 1%SDS, and protease inhibitor cocktail) was used for protein extraction.An ECL system purchased from GE Healthcare (Fairfield, Conn., USA) andstored at 4 and a restore western blot stripping buffer purchased fromThermo Scientific (Rockford, Ill., USA) were used for immunoblotanalysis. Rabbit anti-phospho-AMPK (at Thr172)), AMPK, phospho-FoxO1(Ser256), FoxO1, phospho-FoxO3 (Ser253), FoxO3, HSP27, αB-crystallin,phosphor-S6K (Thr389), S6K, phospho-Akt1 (Ser473), and Akt1 polyclonalantibodies were purchased from Cell Signaling Technology (Beverly,Calif., USA) and used. Rabbit anti-muscle RING-finger protein-1 (MuRF1)and F-Box Only Protein 32 (MAFbx/atrogen) polyclonal antibody werepurchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA) andused, and mouse anti-heat shock proteins (HSP) 90, 72, and 60 werepurchased from Stressgen (Victoria, BC, Canada) and used. Mouseanti-glyceraldehydes-3-phosphate dehydrogenase (GAPDH) antibodies werepurchased from Abcam (Cambridge, UK) and HRP-conjugated anti-mouse IgGand anti-rabbit IgG were purchased from Cell Signaling Technology andused.

2) Cell Culture

C2C12 myoblasts were purchased from American Type Culture Collection(Rockville, Md., USA) and cultured in a DMEM medium containing 4,500mg/L glucose supplemented with 10% fetal bovine serum (Hyclone, Logan,Utah, USA) and 1% antibiotics/antimycotics Gibco, Burlington, Ontario,Canada). The myoblasts were stored under conditions of 37° C. and 5%CO₂. The myoblasts were grown on a 6-well culture plate for immunoblotanalysis and measurement of diameters of myotubes. The myoblasts weremaintained in each well for 5 days by replacing the medium with adifferentiation medium (DMEM containing 2% horse serum and 1%antibiotics/antimycotics) at about 80% confluent state and induced to bedifferentiated into myotubes. The medium was replaced with a new mediumevery two days.

3) Measurement of Cell Size

To verify the effect of T1AM on the size of C2C12 myotubes, the cellswere fixed with 4% paraformaldehyde and photographed at 200×magnification on an Axiovert 200 optical microscope. For analysis, thecells were divided into 9 fractions in order to randomly select thecells. The diameter of each myotube was measured using Image J software(NIH, Frederick, Md., USA).

4) Immunoblot Analysis

The cells were obtained with a RIPA buffer, degraded by repeated suctionthrough a 21 gauge needle, and the transferred to a 1.5 mL microtube. Asample was cultured on ice for 5 minutes and centrifuged at 13,000 rpmat 4° C. for 10 minutes. A supernatant was obtained with whole-cellsoluble lysates and the protein concentration was determined throughBradford assay. To detect AMPK, phospho-AMPK (p-AMPK), FoxO1, p-FoxO1,FoxO3, p-FoxO3, Akt1, p-Akt1, S6K, p-S6K, MuRF1, MAFbx, HSP90, HSP72,HSP60, HSP27, αB-crystallin, and GAPDH, a total of 30 μg of proteins waselectrophoresed on 8 to 10% SDS-PAGE.

The proteins were electrophoretically transferred from the gel to anitrocellulose membrane. The membrane reacted with a blocking buffer (1XTBS, 0.5% Tween-20 with 5% w/v nonfat dry milk) for 1 hour at roomtemperature and then washed with 10 mL TBST three times every 10minutes. Thereafter, the membranes reacted with a primary antibodydiluted appropriately with 10 mL TBST (1:500 to 1:10,000) overnight at4° C. The membrane reacted with a HRP-conjugated secondary antibody fordetection of bound proteins in 10 mL TBST at room temperature for 1 hourby stirring and then washed with 10 mL TBST three times every 10minutes. An immunocomplex was detected by the ECL system (GE Healthcare,Fairfield, Conn., USA) and the obtained bands were quantified by ImageJ1.47t software (NIH, MD, USA). The protein density was normalized by thedensity of GAPDH. To detect the GAPDH, the membrane was washed with TBSTthree times for every 10 minutes and then cultured in a restore bufferfor 30 minutes at room temperature to be stripped.

5) Immunofluorescence and Confocal Microscope

The cells on each 6-well plate were washed three times with 1× PBS andfixed with 4% paraformaldehyde for 30 minutes at room temperature.Thereafter, the cells were then treated with 0.2% Tritin X-100 for 10minutes on ice to ensure permeability and blocked from the 1× PBS with3% BSA. The cells were stained with primary antibodies against FoxO1 andFoxO3 diluted at 1:100 in 1× PBS, respectively, and reacted with Alexa488-conjugated secondary antibody diluted at 1:1,000. Finally, the cellswere washed three times with 1× PBS and then a mounting mediumcontaining DAPI (Vector Laboratories, Burlingame, Calif., USA) wasdropped on the cells. Fluorescent-labeled cells were detected with aCarl Zeiss LSM750 confocal microscope (Jena, Germany).

6) Analysis of Activity of 26S Proteasome

Two groups of myotubes were trypsinized and then washed with a freshdifferentiation medium. Among three determinants of proteasomeactivities (trypsin-, chemotrypsin- and caspase-like activities), thechemotrypsin-like activity is regarded as representative of the proteasecapacity of the proteasome. The chemotrypsin-like activity wasdetermined according to the manufacturer's protocol using a PromegaProteasome-Glo cell-based luminescence assay kit (Promega, Madison,Wis., USA) by approximately 7,500 cells measured by a cell counter(Biorad, Hercules, Calif., USA) in 50 μl of the differentiation medium.To confirm the specificity of the analysis, a partial sample containingthe same number of cells was pretreated with a proteasome inhibitor,epoxomicin, at a concentration of 10 μM for 30 minutes. Thechemotrypsin-like activity was measured by the same process and theresult was used as a background signal for analysis. The luminescencewas measured with a GloMax 20/20 Luminometer (Promega).

7) Statistical Analysis

All values corresponding to the measurement result were represented bymean±SEM. A difference between the groups in means for biochemicalmeasurement (e.g., AMPK, Akt1, etc.) was verified by an independentsample t-test. Statistical analysis was performed using SPSS/PC+, andsignificance was determined at P=0.05.

2. Experimental Result 1) Muscular Atrophy Effect of T1AM in MuscleCells

To determine whether T1AM induced muscular atrophy in C2C12 myotubes,cells were photographed under a phase contrast microscope (FIG. 1A) andthe diameter was measured at 200× magnification (FIG. 1B).

As a result, as shown in FIGS. 1A-1B, it was shown that when 75 μM ofT1AM was treated for 6 hours, the size of myotube was decreased by 0.13times as compared to a vehicle control (16.97±0.32 m).

2) Increase in AMPK Phosphorylation in T1AM-Treated Cells

As shown in FIGS. 2A-2C, it was shown that the AMPK phosphorylation wasremarkably increased (2.7 times) in the T1AM-treated group compared tothe control in the immunoblotting analysis, whereas the total expressionlevels of AMPK were similar between the two groups. As a result, theexpression ratio of p-AMPK/AMPK in the T1AM-treated group was 2.0 timeshigher than that of the control (FIG. 2C).

3) Down-Regulation Of Anabolic Signaling Activity in T1AM-Treated Cells

As shown in FIGS. 3A-3E, it was shown that a phosphorylation level ofAkt1 was significantly down-regulated in the T1AM-treated group comparedto the control, but the non-phosphorylation level between the two groupswas similar to each other. Accordingly, the expression ratio ofp-Akt1/Akt1 in the T1AM-treated group was 0.45 times lower than that ofthe control (FIG. 3C). Further, the p-S6K level was lowered by T1AMtreatment and as a result, the expression ratio of p-S6K/S6K in theT1AM-treated group was 0.53 times lower than that of the control (FIG.3E).

4) Down-Regulation of p-FoxO1 and p-FoxO3 in T1AM Treated Cells

As shown in FIGS. 4A-4F, it was shown that the total expression of FoxO1in the T1AM-treated group was 2.5 times higher than that of the control(in Ser256), whereas the phosphorylation level between the two groupswas similar. It was shown that the expression ratio of p-FoxO1/FoxO1 was0.66 times lowered in the T1AM-treated group (FIG. 4D). On the otherhand, it was shown that the total expression of FoxO3 was not differentbetween the T1AM-treated group and the control, but the p-FoxO3 levelwas 0.58 times lowered in the T1AM-treated group. Thus, the expressionratio of p-FoxO3/FoxO3 in the T1AM-treated group was 0.39 times lowerthan of the control (FIG. 4F).

5) Up-Regulation of MuRF1 Expression and Proteasome Activity

As shown in FIGS. 5A-5D, among experimented catabolic signaling markers,the expression of MuRF1 in the T1AM-treated group was 1.8 times higherthan that in the control (FIGS. 5A and 5B), whereas the expression ofMAFbx was not affected by the T1AM treatment (FIGS. 5A and 5C). Thechymotrypsin-like activity, one of the major catabolic properties ofproteasome, in the T1AM-treated group was 1.5 times higher than that ofthe control (FIG. 5D).

6) Decrease in Expression of HSP72 and αB-crystallin in T1AM TreatedCells

As shown in FIGS. 6A-6D, it was shown that the expression levels ofHSP72 and αB-crystallin in the T1AM-treated group were 0.89 times and0.63 times down-regulated compared to the control, whereas a differencein HSP60 expression between the two groups was not statisticallysignificant.

3. Conclusion

The activity of FoxOs is known to be regulated by an antagonistic effectof AMPK and Akt1. That is, a decrease in expression ratio of p-FoxO/FoxOcorresponds to up-regulated p-AMPK and corresponds to down-regulatedp-Akt1. This induces protein degradation as one of the catabolism. Asseen from the above experimental results, AMPK, FoxO1, FoxO3, MuRF1 andproteasome involved in the muscle protein degradation mechanism areactivated by T1AM mediated hypometabolism, whereas AKT1, S6K, heat shockprotein 72 (HSP72), and αB-crystallin involved in the muscle proteinsynthesis mechanism are inactivated. Therefore, thehypometabolism-inducing substance according to the present invention,particularly T1AM, induces hypometabolism to inhibit energy metabolismand activating the catabolism, thereby activating the protein associatedwith the muscle protein degradation mechanism and inhibiting theproteins associated with the muscle protein synthesis mechanism, and asa result, the sizes of the myoblasts are decreased.

What is claimed is:
 1. A screening method for a drug for treatingmuscular atrophy, comprising: (a) inducing muscular atrophy byadministering to a normal cell or a normal animal ahypometabolism-inducing substance selected from the group consisting of3-iodothyronamine (T1AM), [D-Ala2, D-Leu5] enkephalin (DADLE),5′-adenosine monophosphate (5′-AMP), and hydrogen sulfide (H₂S); (b)treating a candidate substance in the cell or animal treated with thehypometabolism-inducing substance; (c) evaluating the degree ofimprovement or treatment of muscular atrophy in the cell or animaltreated with the candidate substance; and (d) determining the candidatesubstance as a drug for treating muscular atrophy.
 2. The screeningmethod of claim 1, wherein in step (d), as compared to a control treatedwith DMSO, physiological saline, sterilized distilled water,carboxymethyl cellulose or phosphate buffered saline (PBS), when thegroup treated with the candidate substance has at least one resultselected from the group consisting of: an increase in size of myotubesor size of muscle; a decrease in expression ratio of p-AMPK/AMPK; anincrease in expression ratio of p-Akt1/Akt1; an increase in expressionratio of p-S6K/S6K; an increase in expression ratio of p-FoxO1/FoxO1; anincrease in expression ratio of p-FoxO3/FoxO3; a decrease in expressionlevel of MuRF1; a decrease in activity of proteasome; an increase inexpression level of heat shock protein 72 (HSP72); and an increase inexpression level of αB-crystallin, the candidate substance is determinedas the drug for treating muscular atrophy.
 3. The screening method ofclaim 1, wherein the hypometabolism-inducing substance in step(a) is3-iodothyronamine (T1AM).
 4. The screening method of claim 3, wherein adose of the 3-iodothyronamine (T1AM) is 0.1 μM to 1000 μM in the case ofthe cell, and 10 to 500 mg/kg per unit weigh (kg) of the animal in thecase of the animal.
 5. The screening method of claim 1, wherein theadministering of the hypometabolism-inducing substance is performed by amethod selected from the group consisting of oral administration,intraperitoneal administration, intravenous administration,intramuscular administration, subcutaneous administration, andintradermal administration.
 6. The screening method of claim 1, whereinthe cell is a muscle cell and, the animal is a vertebrate.